Downregulation of UBE4B promotes CNS axon regrowth and functional recovery after stroke

Summary The limited intrinsic regrowth capacity of corticospinal axons impedes functional recovery after cortical stroke. Although the mammalian target of rapamycin (mTOR) and p53 pathways have been identified as the key intrinsic pathways regulating CNS axon regrowth, little is known about the key upstream regulatory mechanism by which these two major pathways control CNS axon regrowth. By screening genes that regulate ubiquitin-mediated degradation of the p53 proteins in mice, we found that ubiquitination factor E4B (UBE4B) represses axonal regrowth in retinal ganglion cells and corticospinal neurons. We found that axonal regrowth induced by UBE4B depletion depended on the cooperative activation of p53 and mTOR. Importantly, overexpression of UbV.E4B, a competitive inhibitor of UBE4B, in corticospinal neurons promoted corticospinal axon sprouting and facilitated the recovery of corticospinal axon-dependent function in a cortical stroke model. Thus, our findings provide a translatable strategy for restoring corticospinal tract-dependent functions after cortical stroke.


INTRODUCTION
Promoting the regrowth of corticospinal tract (CST) axons to reinnervate the spinal cord is a promising strategy for restoring lost functions after cortical stroke. 1,2 However, CST axons in the adult CNS are intrinsically refractory to regrowth after cortical stroke. An understanding of the basic biological processes within neurons that intrinsically inhibit axonal regrowth is a prerequisite for developing interventions for stroke or CNS injury. In the past decade, numerous intrinsic factors that regulate the regrowth capacity of CNS axons have been identified. [3][4][5][6][7] Studies have shown that increasing the activity of mammalian target of rapamycin (mTOR) markedly augments the axonal regeneration capacity of injured neurons. 4,5,[8][9][10] Moreover, the p53 pathway has been reported to regulate the intrinsic axonal regrowth capacity of CNS neurons. 11,12 However, little is known about the key upstream regulatory mechanism by which the p53 pathway and mTOR pathway control CNS axon regrowth.
Ubiquitin pathways, which participate in the protein quality control system, are responsible for maintaining cellular homeostasis by degrading unfolded and misfolded proteins. After axonal injury, damaged proteins must be quickly eliminated to help restore cellular homeostasis for axonal regrowth. Therefore, ubiquitin pathways have been suggested to play important roles in this process. 13,14 However, how and to what extent ubiquitin pathways regulate CNS axon regrowth remain unclear. Because ubiquitin pathways have been reported to regulate axonal regrowth through the p53 axis, 11 we speculate that ubiquitin-mediated degradation of p53 might play an important role in controlling CNS axon regrowth.
We screened a series of genes involved in regulating ubiquitination of p53 to test this hypothesis and identified the ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase, as a new repressor of CNS axon regrowth. Surprisingly, we found that UBE4B also played a key role in modulating the mTOR pathway, another major regeneration-promoting pathway, 4,15 via its substrate KLHL22. In addition, overexpression of the ubiquitin variant UbV.E4B, which was previously designed as an effective inhibitor of UBE4B activity, 16 in corticospinal neurons led to robust CST axon sprouting and the recovery of CST axon-dependent functions in a rat cortical stroke model. Thus, our findings not only reveal the key mechanism that

Identification of UBE4B as a key inhibitor of CNS axon regrowth
We screened a series of ubiquitin-related genes involved in p53 degradation, including HAUSP, Pirh2, COP1, MDM4, and UBE4B, to identify the key ubiquitin-related molecule that regulates axonal regrowth via the p53 pathway. 11,[17][18][19][20][21] In a pilot experiment, we employed an optic nerve crush (ONC) model to investigate the roles of these genes in regulating CNS axon regeneration because the anatomical structure of the optic nerve is relatively simple and because findings obtained using optic nerve injury models have been verified in other models of axonal injury. 6 Adeno-associated virus (AAV) serotype 2 (AAV2) vectors carrying single-guide RNA (sgRNA) and cyclization recombination enzyme (Cre) were intravitreally injected into the retinas of Rosa26-Loxp-Stop-Loxp-Cas9 knockin mice (LSL-Cas9 mice) to delete the target genes via CRISPR/Cas9 technology, 22,23 and the gene knockout efficiency obtained using this method was validated (Tables S1 and S2). We also coadministered AAV2-sgControl and AAV2-Cre into control LSL-Cas9 mice by intravitreal injection. Two weeks after virus injection, ONC was performed using a well-established protocol. 4 After another 2 weeks, axon regeneration was evaluated by injecting the anterograde fluorescent tracer-conjugated cholera toxin subunit B (CTB) into the vitreous humor 2 days prior to tissue collection ( Figure 1A).
As a result, knockout of MDM4 in retinal ganglion cells (RGCs) promoted optic nerve axon regeneration to some extent, i.e., to approximately the same extent reported in a previous study ( Figure 1B). 11 Interestingly, knockout of COP1 also promoted axonal regeneration to some extent, promoting regeneration of a limited number of axons over a limited distance ( Figure 1B). However, after knockout of other factors, such as Pirh2 and HAUSP, which have also been proven to be involved in ubiquitin pathways, almost no or few regenerating axons were observed after injury, similar to results observed in control mice ( Figures 1B and 1C), although these proteins are expressed in the adult CNS (proteinatlas.org). [24][25][26][27][28] In addition, RGC survival was not affected by knockout of these genes individually (data not shown). Besides, we found the deletion of MDM2, a previously reported gene, 11 leads to some axon regeneration after optic nerve injury (data not shown). Interestingly, we found that only UBE4B knockout promoted abundant RGC axon regeneration after injury and that the regenerated axons extended approximately 1.5 mm from the lesion ( Figure 1B), possibly because UBE4B knockout leads to the activation of other pathways mediating axon regeneration in addition to the p53 pathway. Moreover, we knocked out both MDM2 and UBE4B in UBE4B f/f -LSL-Cas9 mice via intravitreal injections of AAV2-Cre and AAV2-U6-sgMDM2. Interestingly, the deletion of both UBE4B and MDM2 leads to more robust regeneration than the deletion of UBE4B or MDM2 alone without affecting the survival rate of RGCs (data not shown).
Given the limited knockout efficiency and potential off-target effects of the CRISPR/Cas9 technique, we generated homozygous conditional UBE4B-knockout (UBE4B f/f ) mice to further verify the effect of UBE4B knockout on axonal regeneration (Figure 2A). AAV2-Cre or AAV2-PLAP control was intravitreally injected into UBE4B f/f mice, and the regeneration of optic nerve axons was significantly increased in the group in which UBE4B was deleted in RGCs compared with the control group (Figures 2A-2C), consistent with the results obtained using the CRISPR/Cas9 technique. Because gene knockout might also affect RGC survival, we assessed the RGC survival rate 2 weeks after ONC injury in AAV2-PLAP-injected and UBE4Bknockout mice with the pan-RGC marker RNA binding protein with multiple splicing (RBPMS) 29 and retinal whole-mount staining for Tuj1. 4,9 The RGC survival rate was not affected by UBE4B knockout (Figures 2D,  2E, S1A, and S1B), indicating that UBE4B knockout promotes optic nerve regeneration by enhancing the regeneration potential of surviving RGCs after ONC rather than by protecting against RGC death after ONC.
UBE4B deletion promotes optic nerve regeneration via both the p53 and mTOR pathways As heterozygosity of UBE4B is sufficient to induce cellular stress in at least some types of neurons 30 and that stress signaling is an important component of the regenerative response, 31 we first sought to investigate whether UBE4B knockout primes RGCs for more robust axon regeneration by activating stress response pathways prior to injury. Immunohistochemical staining for the transcription factors ATF3 and c-Jun was conducted to clarify whether this possible mechanism might contribute to the regenerative phenotypes observed in UBE4B-knockout animals ( Figures S2A and S2C 32 Interestingly, a previous study showed that the KLHL22 ubiquitin ligase complex plays a conserved role in mediating the activation of mTORC1 and downstream events in mammals, and the KLHL22/mTOR axis was proven to activate amino iScience Article acid-dependent mTORC1 signaling. 33 Therefore, we speculated that UBE4B deletion might increase the regeneration potential of RGCs via the KLHL22/mTOR pathway. We directly investigated the contributions of p53-or KLHL22-dependent pathways to the effects of UBE4B deletion on axonal regeneration by crossing UBE4B f/f mice with LSL-Cas9 mice to generate homogeneous mutants carrying two floxed genes (UBE4B and Cas9). The double-mutant mice received an intravitreal viral injection of AAV2-Cre and/or AAV2-sgRNA to induce the deletion of one or both genes in RGCs ( Figure 4A). The regeneration of injured optic nerve axons was not significantly different between the p53-or KLHL22knockout mice and the control mice ( Figures 4B and 4C). However, additional knockout of p53 or KLHL22 led to a significant decrease in axonal regeneration in UBE4B-knockout mice ( Figures 4B and 4C), indicating that p53 and KLHL22 deletion partially inhibited axonal regeneration triggered by UBE4B deletion. Moreover, no significant differences in neuronal survival were observed among the UBE4B-knockout, p53-knockout, KLHL22-knockout groups, and the double-knockout groups ( Figures S3A and S3B), suggesting that the apoptosis of RGCs does not impair axonal regeneration. Furthermore, combining AAV2-Cre and AAV2-sgRNA (for p53 or KLHL22) with AAV expressing ciliary neurotrophic factor (AAV-CNTF) did not affect axonal regeneration induced by CNTF ( Figures S4A-S4C). P53 knockout and KLHL22 knockout did not affect the survival rate of RGCs promoted by CNTF overexpression after injury ( Figures S4D and  S4E). Conclusively, we confirmed that UBE4B deletion in RGCs dramatically promoted axonal regeneration following optic nerve injury through a mechanism depending on the p53 and KLHL22/mTOR pathways ( Figure 4D).

OPEN ACCESS
Previous studies have indicated that modulation of the PTEN/mTOR pathway increases the survival of RGCs and promotes axon regeneration. 4,5,34,35 Our finding that the effect of UBE4B deletion also involves the mTOR pathway motivated us to explore the synergistic effect of UBE4B and PTEN deletion on axonal regeneration. In Rosa26-LSL-Cas9 mice, we deleted PTEN using AAV2-Cre mixed with AAV2-sgPTEN (PTEN-knockout group). In UBE4B f/f -LSL-Cas9 mice, we knocked out both PTEN and UBE4B by administering intravitreal injections of AAV2-Cre and AAV2-U6-sgPTEN (UBE4B/PTEN-knockout group) (Figure 5A). We found that double knockout of UBE4B and PTEN induced optic nerve regeneration to a greater extent than PTEN knockout alone (Figures 5B, 5a, 5b and 5c). Specifically, compared with UBE4B knockout alone and PTEN knockout alone, knockout of both UBE4B and PTEN induced more axonal regeneration at 500, 750, 1,000, 1,500, 2,000, 2,500, 3,000, and 3,500 mm from the injury site, with many axons reaching the optic chiasm within 2 weeks after injury, which was rare after knockout of either gene alone (Figures 2C and  5C). Notably, the survival of RBPMS + RGCs increased to approximately 50% in UBE4B/PTEN doubleknockout mice ( Figures 5D and 5E). Based on these results, the deletion of both UBE4B and PTEN led to more robust regeneration than the deletion of UBE4B or PTEN alone, possibly because UBE4B knockout promoted the activation of the p53 pathway in addition to the mTOR pathway, further increasing the axonal regeneration capacity of RGCs after ONC injury.

UBE4B knockout promotes CST axon sprouting in the spinal cord after unilateral cortical stroke
After verifying that UBE4B deletion in RGCs promotes RGC axon regeneration after injury, we attempted to assess whether this phenomenon also occurred in cortical neurons. Before the CST axon regrowth study, we first sought to assess whether the molecular pathways found to be involved in axonal regeneration in RGCs were also involved in axon regeneration in UBE4B-knockout corticospinal neurons. Therefore, we crossed UBE4B f/f mice with UBE4B f/f and Camk2a-Cre/+ mice to generate double-mutant mice in which UBE4B was specifically knocked out in neurons expressing Camk2a ( Figure S5A). Immunoblot analyses of these cortical neurons confirmed that UBE4B deletion upregulated the expression of p53, KLHL22, pS6, and mTOR (Figures S5B and S5C), consistent with the alterations in RGCs induced by UBE4B knockout. This result confirmed that UBE4B knockout also regulated both the p53 pathway and mTOR pathway in cortical neurons.
We next sought to assess whether UBE4B deletion promoted CST axon regrowth in a cortical stroke model. We first injected AAV9-Cre (UBE4B-knockout group) or AAV9-PLAP (control) into the right sensorimotor cortices of neonatal UBE4B f/f mice ( Figures S6A and S6B). Six weeks after the virus injection, unilateral cortical stroke in the left sensorimotor cortex was elicited by generating a minimally invasive and reproducible photochemical cortical lesion to disrupt CST axons ( Figures S6A and S6B). Six weeks after unilateral cortical stroke, AAV9-mCherry was injected into the intact sensorimotor cortices of the mice, and axons in the intact sensorimotor cortex were labeled with mCherry before sacrifice. The difference in injuryinduced CST axon sprouting between control and UBE4B-knockout mice was evaluated by constructing color-coded heatmaps showing the density of CST sprouting in the contralateral denervated spinal hemicord. Although limited sprouting was observed in the control mice, the sensorimotor cortices of UBE4Bknockout mice showed increased sprouting into the ipsilateral spinal cord at the cervical level in Mid, Z1, and Z2 ( Figure S6C). More axons extended into D1, D2, and D3 through the midline in the UBE4Bknockout mice ( Figure S6D). Thus, the deletion of UBE4B strikingly enhanced the sprouting of adult CST axons following cortical stroke.
Overexpression of UbV.E4B in corticospinal neurons improves functional recovery in a rat cortical stroke model by promoting CST axon sprouting The UBE4B knockout-induced increase in CST axon sprouting inspired us to assess whether this axonal plasticity contributed to functional recovery after cortical stroke. 36 Because the clinical application of iScience Article UBE4B knockout strategies is limited, we sought to overexpress UbV.E4B, a ubiquitin variant-based inhibitor of UBE4B, and subsequently inhibit the activity of UBE4B (the sequences of UbV.E4B and UbiquitinWT are provided in Table S3). 16 Before this experiment, we first attempted to test the effect of UbV.E4B overexpression in RGCs on optic nerve axon regrowth. For this experiment, we intravitreally injected AAV2-PLAP, AAV2-HA-UbiquitinWT, or AAV2-HA-UbV.E4B into the wild-type mice ( Figure S7A). Unlike the overexpression of HA-UbiquitinWT or PLAP, overexpression of UbV.E4B in mature RGCs promoted robust optic nerve regeneration after ONC ( Figures S7B-S7D). We next examined whether UbV.E4B functioned in RGCs. As UBE4B promotes p53 polyubiquitination and degradation, 19 we used immunohistochemistry to analyze the expression of p53 in the RGC layer of retinal sections. Our data showed that HA-labeled UbV.E4B was overexpressed ( Figures S7D and S7E) and reduced the degradation of p53 in RGCs ( Figures S7F and S7G), indicating the inhibitory effect of UbV.E4B on UBE4B.
After confirming the ability of UbV.E4B overexpression to promote optic nerve regeneration after ONC, we next sought to assess whether this strategy also promoted CST axon regrowth and related functional iScience Article recovery after cortical stroke. For this experiment, adult rats were first trained to master a single-pellet grasping task and irregular ladder walking task ( Figure 6A) as the CST is known to be crucial for dexterous movements. 1,37 Then, we disrupted CST axons on one side by inducing cortical stroke, and both triphenyltetrazolium chloride (TTC) staining (1 day after stroke) and protein kinase C gamma (PKCg) staining in the iScience Article cervical spinal cord (12 weeks after stroke) were conducted to validate the damage to the sensorimotor cortex ( Figures S8A-S8C). We detected approximately 10% spared CST axons from the impacted cortex compared to the uninjured side ( Figure S8D), suggesting that our rat cortical stroke model resulted in consistent lesions of the sensorimotor cortex. AAV9-HA-UbV.E4B was injected into the sensorimotor cortex on the intact side 3 days after stroke ( Figure 6A). AAV9-UbiqutinWT was injected as a control ( Figure 6B). The difference in injury-induced sprouting between control-and UbV.E4B-treated rats was evaluated by constructing color-coded heatmaps showing the density of CST sprouting in the contralateral denervated spinal hemicord ( Figure 6C). We found that HA-UbV.E4B was abundantly expressed in the cortex ( Figures S9A and S9B), sprouting into the ipsilateral spinal cord was increased at the cervical level in the sensorimotor cortices of UbV.E4B-treated rats, and more axons extended into the contralateral denervated spinal cord through the midline (Figures 6C and 6D). In other words, sprouting of CST axons into the ipsilateral spinal cord was markedly increased in UbV.E4B-treated rats, whereas limited sprouting was observed in the controls (Figures 6C and 6D). By analyzing the video recordings of the behavioral performances of rats in a double-blinded manner, we found that rats treated with UbV.E4B exhibited significant recovery of forelimb function in both tasks ( Figures 6E and 6G), indicating that CST axon regrowth contributed to the improvement of fine forelimb motor functions after stroke. More specifically, UbV.E4B treatment significantly increased the overall success rate in the pellet retrieval test ( Figure 6E). According to scores assigned based on the Eshkol-Wachman Movement Notation (EWMN) system, improvements in the grasping component of the pellet retrieval test, but not the reaching or retrieval component, mainly contributed to the observed functional recovery of the forelimb ( Figure 6F). Moreover, although the reaching trajectories of the rats were highly variable after stroke ( Figure S10A), a significant difference in trajectory variability was not observed compared with that in the ubiquitin-treated group ( Figure S10C). The endpoint distributions also showed no obvious differences among groups (Figures S10B and S10D), further verifying that improvements in grasping, but not improvements in reaching, primarily contributed to the observed functional recovery in the pellet retrieval test.

Ablation of sprouting CST axons abolishes the recovery of skilled locomotor ability
We selectively ablated sprouting axons in the cervical spinal cord by administering an intraperitoneal (i.p.) injection of diphtheria toxoid (DT) after a unilateral injection of AAV2/Retro-Cre into the denervated side of the cervical spinal cord (C5-C7) and an injection of AAV9-Flex-DTR (ablation) or AAV9-Flex-PLAP (control) into the intact side of the cortex in a set of rats expressing UbV.E4B, as described above, to further verify the contribution of CST sprouting in the cervical spinal cord to functional recovery ( Figures 7A and 7B). We verified that the behavioral performance of the rats was not altered by these intraspinal and cortical injections ( Figures 7C and 7D). However, 2 weeks after DT administration, the improved performance of rats expressing UbV.E4B on the single-pellet retrieval task and irregular ladder walking of the denervated forelimb was remarkably abolished. On the other hand, a significant difference in the performance of the intact forelimb on irregular ladder walking was not observed (Figures 7C and 7D). Consistently, ablation of CST axons was observed in the cervical spinal cord on the denervated side ( Figures 7E and 7F). Thus, our results suggested that axonal sprouting in the spinal cord is required for the recovery of skilled motor ability after unilateral photothrombotic stroke.

DISCUSSION
Using in vivo CRISPR screening and gene manipulation strategies, we found that UBE4B simultaneously modulates two of major pathways that control optic nerve and CST axon regrowth, i.e., the p53 and KLHL22/mTOR pathways. We developed an AAV-based method for overexpressing UbV.E4B that mimics iScience Article axonal regrowth triggered by UBE4B deletion to translate this finding into a potential translational strategy for restoring CST-dependent functions after cortical stroke in clinical applications.
Surprisingly, we found that among molecules involved in ubiquitin pathways, UBE4B, but not MDM4, played a major role in regulating CNS axon regrowth. A previous study reported that conditional deletion of MDM4 promotes axon regeneration. 11 In our study, we observed little regrowth of optic nerve axons in mice with MDM4 deletion, but UBE4B deletion induced much more robust axon regeneration after ONC injury. Although MDM4 might also play a role in regulating axonal regrowth via the p53 pathway, UBE4B activates the KLHL22/mTOR pathway in addition to the p53 pathway. This finding explains why UBE4B deletion exerted a superior effect on promoting axonal regrowth and emphasizes the importance of considering multiple pathways when designing interventions for promoting functional recovery through axonal regrowth.
Previously, several strategies, such as activation of mTOR and/or overexpression of p53, have been proposed to promote CNS axon regrowth. 8,[38][39][40] The mTOR signaling pathway has been suggested to play an important role in synaptogenesis and differentiation, particularly axon regeneration and neuronal survival after CNS injury. 38,41 P53, a well-known tumor suppressor, is a multifunctional sensor of a number of cellular signals and pathways essential for angiogenesis, cell metabolism, DNA damage, cell cycle regulation, apoptosis, and nerve regrowth. 39,42,43 However, the key molecule involved in the simultaneous modulation of these two important pathways was not identified previously. In the present study, UBE4B, which has been proven to promote p53 polyubiquitination and degradation and inhibit p53-dependent transactivation and apoptosis, 19 played a major role in CNS axon regrowth. Further analysis showed that UBE4B, in addition to p53, activated the mTOR pathway via its substrate KLHL22 to promote axon regrowth, revealing a new mechanism underlying the regulation of CNS axon regrowth.
In a rat cortical stroke model, we showed that overexpression of UbV.E4B in cortical neurons restored CSTdependent function by promoting CST axon regrowth. Although numerous intrinsic mechanisms underlying the regulation of CNS axon regrowth have been explored, few have been studied in translatable settings. 1,44 Here, in a translatable setting, we showed that overexpression of UbV.E4B alone was sufficient to trigger robust CST axon regrowth and functional recovery, suggesting that UbV.E4B is a potential treatment for cortical stroke. However, regenerating CST axons might need to be fine-tuned to better perform their functions. Because exercise and electrical stimulation promote the plasticity of CST axons toward the development of functional spinal networks, 36,44-47 the combination of electrical stimulation and UbV.E4B overexpression might further improve functional recovery after cortical stroke. Studies examining this combination strategy and the potential of systemic UbV.E4B delivery as a treatment for cortical stroke are ongoing.

Limitations of the study
First, downregulation of UBE4B promotes axon regeneration without enhancing the survival rate of RGCs after injury; whether there are new strategies beneficial to not only RGC survival but also axon regeneration is worth exploring. Second, besides mTOR and p53, other druggable targets for UBE4B might need to be explored in future study because there are numerous substrates for UBE4B. Third, AAV gene delivery is featured by high price and technical difficulty to safely deliver genes to the CNS. Last, regenerating CST axons might need to be fine-tuned by combination of electrical stimulation, UbV.E4B overexpression, and so on, to better reinvent functional spinal networks and perform their functions after cortical stroke.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
Zhejiang University has filed a patent application related to this work, with X.W. listed as an inventor. X.W. is a scientific co-founder of WeQure AI Ltd. All the other authors declare no conflicting interests.

Intravitreal injection and ONC
For intravitreal injection, animals were anesthetized with avertin, and then the edge of the eyelid was clamped with a small artery clamp to expose the conjunctiva. AAV (1-3 mL) was injected intravitreally on postnatal day (P)28, and Alexa Fluor-conjugated CTB (CTB-555, 1 mg/mL; 1-2 mL, Invitrogen) was injected intravitreally at P54. The agents were injected with a fine glass pipette attached to the Hamilton syringe using plastic tubing. The CTB-555 injection was performed 2-3 days before euthanasia to anterogradely trace regenerating RGC axons. Mice with obvious eye inflammation or shrinkage were sacrificed and excluded from further experiments. Two weeks after the virus injection, intraorbital ONC was performed.
After the mice were anesthetized and an incision was made in the conjunctiva, the optic nerve was crushed using a pair of forceps with a 0.1 mm-wide tip for 5 s 1-2 mm behind the optic disk.

Unilateral photothrombotic stroke
Animals were fixed in a stereotactic frame, and the skull was exposed. A cold light source (WeiHaiLiXin, LX-D40, 40 W, 9000 mW/cm 2 ) was positioned over an opaque template with an opening (a 10 mm 3 5 mm rectangle for rats or a circle with a diameter of 2.5 mm for mice) to target the sensorimotor cortex corresponding to the preferred paw. For rats, Rose Bengal (20 mg/kg body weight, 20 mg/mL Rose Bengal in saline) was injected via the tail vein, and after 2 min, the brain was illuminated through the skull for 15 min. For mice, Rose Bengal (10 mg/kg body weight, 5 mg/mL Rose Bengal in saline) was injected via the tail vein 10 min before the brain was illuminated through the intact skull for 15 min.

Virus injection
Neonatal Ube4B f/f mice were cryo-anesthetized for 30 s and 3 mL of either AAV9-Cre or AAV9-PLAP were injected into the right sensorimotor cortex using a 10-mL Hamilton microsyringe with a pulled-glass micropipette (68606, RWD, China). After the injection, the mice were placed on a heating pad and returned to their mothers after they regained normal color and activity. Six weeks later, unilateral photothrombotic stroke was induced in the left sensorimotor cortex. We injected 4 mL of AAV9-mCherry into the sensorimotor cortex 6 weeks after stroke at a rate of 80 nL min À1 (300 nL per site, twelve sites) to anterogradely label the CST. Mice were maintained for an additional 2 weeks before being euthanized. All AAV vectors, including AAV9-mCherry and AAV9-Cre/PLAP, were generated at the Viral Core of Zhejiang University, and their titers were adjusted to 1X10 13 copies per mL for injection. The rats were injected with 3 mL of AAV9-UbiquitinWT/UbV.E4B and AAV9-mCherry into the contralesional cortex at a rate of 150 nL min À1 (150 nL per site, eighteen sites) three days after stroke. The rats were placed on soft bedding on a heated blanket maintained at 37 C until they were fully awake.

Selective ablation of CSNs with axons sprouting into the denervated side of the spinal cord
Rats underwent unilateral photothrombotic stroke at P70 and were treated with AAV9-UbiquitinWT/ UbV.E4B at P73. Fourteen weeks after injury, laminectomy was performed at the cervical level. AAV2/ Retro-Cre (ablation) or AAV2/Retro-PLAP (control) was stereotaxically injected into the denervated side of the cervical (C5-C7) spinal cords of AAV9-UbV.E4B-treated rats using the protocol established by previous literatures. 1,48 AAV9-Flex-DTR was then injected into the unlesioned sensorimotor cortex 3 days after the AAV2/Retro-Cre/PLAP injection. After 2 weeks, the animals were subjected to the irregularly spaced horizontal ladder walking task and/or single-pellet retrieval task to reassess skilled limb movement. Diphtheria toxin was then administered (100 mg/kg, i.p.). The animals were subjected to the horizontal ladder walking task and/or single-pellet retrieval task again at 2 and 4 weeks after diphtheria toxin administration.

Tissue preparation
Anesthetized animals were transcardially perfused with 4% paraformaldehyde (PFA). Dissected tissues were postfixed with 4% PFA overnight and then cryoprotected in 15% and 30% sucrose before being embedded and snap-frozen in OCT. Typically, the optic nerve samples were cut into 10 mm-thick sections, retinal tissues were cut into 20 mm-thick sections, and spinal cord tissues were cut into 25 mm-thick sections. The sections were adhered to room temperature charged microscope slides, dried, and frozen until further processing. Sections were then either washed and mounted with antifade reagent for imaging (for example, CTB labeling of optic nerves) or further processed for immunohistochemistry. Some retinas were dissected in toto after postfixation with PFA, washed with PBS, immunostained, cut radially with scissors to flatten the tissue, and then mounted for imaging.

Staining
Tuj1 staining of whole mounts was performed to determine the number of surviving RGCs two weeks after ONC. The retinas were washed with 1X PBS three times in a 96-well plate and then blocked for one hour in PBS containing 5% donkey serum and 0.3% Triton X-100. After incubation with a Tuj1 primary antibody diluted in PBS supplemented with 3% donkey serum and 0.3% Triton X-100 for 0.5-2 days at 4 C, the retinas were washed three times with PBS and incubated with the secondary antibody for 1-2 h at room temperature. After the tissues were washed with PBS, the retinas were mounted on glass slides, and images were captured under a wide-field fluorescence microscope (VS120, Olympus, Japan). Twelve images of different quarters covering the peripheral and central regions of each retina were captured. An individual who was blinded to the groups counted the number of Tuj1 + RGCs.
Immunohistochemical staining was performed by blocking the sections with 5% normal donkey serum and 0.5% Triton X-100 in PBS and incubating them with primary antibodies overnight at 4 C in blocking solution.
After three washes with PBS, sections were incubated with appropriate secondary antibodies conjugated to fluorescent dyes at room temperature.
For TTC staining, rat brains were harvested 1 day after stroke. The brain was dissected and removed promptly and cut into 1.5 mm cortical slices. Sections were immediately stained in a prewarmed 2% 2,3,5-triphenyltetrazolium chloride (TTC) (T8877, Sigma-Aldrich) solution in saline, pH 7.4, for 10 min at 37 C. Sections were then washed, fixed with 4% PFA overnight at 4 C, and imaged. The area devoid of red staining was the infarct area.

Western blot analysis
The mice were lightly anesthetized with isoflurane and then decapitated. The brain tissues were dissected and then homogenized in RIPA buffer containing a protease inhibitor cocktail. After centrifugation, the protein concentration in the supernatant was quantified using the BCA assay. Equal amounts of total protein were electrophoresed on SDS-polyacrylamide gels. The separated proteins were transferred onto polyvinylidene fluoride membranes at 4 C. The membranes were blocked for 2 h with 5% milk in Tris-buffered saline with Tween-20 (TBST) at room temperature and incubated with the primary antibody at 4 C overnight. After washing, the membranes were incubated with the appropriate HRP-coupled secondary antibody for 1 h. Then, the protein bands were detected using an ECL kit according to the manufacturer's instructions. We also probed the membranes with an antibody against GAPDH (1:1000, ABclonal A19056) to verify equal loading. The density of the immunoblot bands was measured with ImageJ software (NIH, Bethesda, MD).

Microscopy
For some retinal sections and whole-mount retinas, individual fluorescence images were acquired using a wide-field fluorescence microscope (VS120, Olympus, Japan). For nerve and spinal cord sections, images were captured using a confocal laser scanning microscope (A1R, Nikon, Japan) with automated tiling, and Z stacks were projected onto a single plane. The brightness and contrast of the images were adjusted, and pseudocolors were applied for presentation. When images were used for quantification, the imaging and processing parameters were kept constant.

Behavioral tests
Thirty adult female Sprague-Dawley rats (200-250 g, 3-4 months of age) were used for histological and behavioral studies. The rats were trained in the single-pellet grasping task and irregular ladder walking task. After 3-4 weeks of training, the baseline performance of each rat was recorded, and only animals that achieved an 80% success rate in the single-pellet grasping task and a 25% error rate in the irregular ladder walking task were included in further experiments. Then, video recordings of the behavioral performances of rats were analyzed in a double-blinded manner. iScience Article Single-pellet grasping task Each rat was placed in a chamber (45 cm 3 13 cm x 40 cm) and allowed to reach for and grasp a pellet (dustless precision pellet, 45 mg, Bioserv) on a shelf through a wide silt in the front of the chamber. The rats were food-restricted to maintain a weight greater than 90% of the free feeding weight throughout the training session. During the test session, 20 pellets were provided within 10 min. The success rate was calculated as the total score/20, where the score was determined according to the criteria listed below. A score of 1 was assigned if the rat directly retrieved the pellet and brought it to its mouth. A score of 0.5 was assigned if the rat successfully grasped the pellet but dropped the pellet inside the box. A score of 0 was assigned if the rat missed the pellet or knocked the pellet off the shelf. All test sessions were videotaped (60 fps) and analyzed further. For a detailed analysis of motor components, scoring based on the EWMN system was performed as described in the original study. 49

Irregular ladder walking task
Rats walked through a ladder, the beams of which were unequally spaced from 1 cm to 5 cm. In each test session, the rats were allowed to walk through the ladder 3 times and videotaped (60 fps) for scoring. The error rate was calculated as the number of error steps/total number of steps. Two types of error steps occurred: 1) Miss: when crossing the ladder, the forelimb either completely missed the rung, or the rung was contacted by the wrist instead of the paw and 2) Slip: when crossing the ladder, the rat placed a few digits instead of the paw on the rungs, causing a subsequent slip. A correct step was defined as precise placement of the center of the palm on the rung and closing of the digits.

Quantification of axon regrowth
First, the experimenters were blinded to the conditions when performing the measurements. The optic nerve was dissected carefully, and longitudinal sections of optic nerves were cryosectioned (section thickness: 10 mm) to quantify the regeneration of axons traced with fluorescent CTB after ONC. Serial optic nerve sections were imaged under a confocal microscope (A1R, Nikon, Japan; 20X objective) using the CTB channel. Images of the nerve sections were exported and viewed in ImageJ software. CTB + axons along the optic nerve were counted at multiple distances (250 mm, 500 mm, 1000 mm, 1500 mm, 2000 mm, etc.) in the anterograde direction from the crush site. At least three images per optic nerve were analyzed. The counts were transformed into axonal density and then multiplied by the approximate cross-sectional area of the nerve (estimated diameter = 250 mm) to estimate the total number of axons on each respective nerve. The following formula was used to calculate the estimated number of regenerated axons at different distances from the crush site on each optic nerve: ad = pr2 * (axon numbers/mm)/[10 mm*(nerve width at the counting site)]. The average estimate for each optic nerve was recorded as a single value for statistical analysis.
A horizontal line was first drawn through the central canal and across the lateral rim of the gray matter, and fibers crossing the spinal cord midline were counted at 20x magnification to quantify the number of sprouting axons. Four vertical lines (main tract, D1, D2 and D3) were drawn from the central canal to the lateral rim to divide the horizontal line into three equal parts. Although the main tract was used to identify midline crossing fibers, D1, D2 and D3 were used to identify sprouting fibers at different distances from the midline. Only fibers that crossed the four lines were counted in each section. The number of sprouting fibers normalized to the number of CST fibers at the medulla level is presented. Images were imported into and analyzed using Python to erode all CST axons to a single pixel width and determine the distribution of sprouted CST axons. The total number of pixels in a particular area thus corresponded to the overall density of the CST label in a specific area of the section. The pixelated data were further processed using Python to generate heatmaps, with red representing the highest axonal density, blue representing the lowest axonal density and white representing the background of the images. For correlation analyses between regenerated CST axons and the success rate in the single-pellet retrieval test, the average number of axons in each animal was plotted.

Quantification of RGC bodies
For quantification of RGCs, eyeballs were cryosectioned at a thickness of 20 mm. The sections were stained for RBPMS (a selective marker of ganglion cells in the mammalian retina) to identify RGCs. Fluorescence images were acquired with a 10x objective using a fluorescence microscope (VS120, Olympus, Japan). At least three sections near the maximum diameter of the eyeball per retina were quantified. In intact ll OPEN ACCESS iScience Article